[Histonet] Section thickness in immunofluorecsence studies

From:

"George Cole"

HISTOTECHS DOING IMMUNOFLUORESCENCE
SSUDIES.

I just visited the hospital I retired from. In talking with
the great gal who replaced me and my good friend who runs the immuno lab I mentioned that when I did immunofluorescence
studies on kidney, I cut sections thicker than anyone else---I was shaky with 2 or 3 micron
sections as a beginner, so I cut one 3 micron, then went up to 5 microns,and, because I had been doing nothing
but muscles and nerves in which I did 8 and 10 micron sections, I included an 8
micron section. Well the kidney turned out to fluoresce with two preps. The 3
micron section was not very dramatic.The 8 micron section was brilliant, but a bit globby;
but to my surprise, all hands chose the 6 micron section.Also, as I had developed the multiple
sections techniques on muscles and nerves, I cut one 3 micron section for slide
1, 2 and 3, then went back and cut a 6 micron section for slide 1, 2 and 3, and
did the same for 8 microns sections. Well, I never went back to the 2 or 3
micron sections. I made 6 microns standard----and got many beautiful photos
brilliantly shining. The 8 micron section was a little much, so I used that only
with bigger pieces of tissue. But remember what we are doing in this
procedure----we are NOT staining fine nuclear detail that takes thin sections
to do justice to---we are staining BLOBS and the photos of the
6 microns sections use to be
passed around getting OOOHHED and AAAHHHED by the residents. Also, about the
multiple sections---this wasn’t always possible, because the kidney
biopsies are usually quite small and I had to leave tissue enough to do the
Jones silvers----but in this case, of the three sections on
one positive prep, only one of the sections lit up. And what did that
tell me?It told me that a single
section per slide work up on this case could miss being positive something like
66% of the time.So I kept doing a
3 micron and as many 6 micron sections cut for each slide as the size of the
tissue would allow. The 3 micron section NEVER was positive, by chance or by
whatever----and I passed around many brilliantly lighted kidney sections photos
of the 6 micron sections.